Pain is defined to have both a perception and physical component. Opioids are still on of the most common analgesics used. Opioids, however, elicit unwanted side effects including respiratory depression, tolerance, and dependence with chronic pain becoming increasingly more prevalent, there is a growing need for novel therapies. By understanding how tolerance develops new approaches to treating pain can be evaluated. The project proosed will evaluate cellular mechanisms of tolerance. More specifically, the role the carboxyl tail of the mu-opioid receptor plays in regulating the receptor will be studies. Phosphorylation of the mu-opioid receptor is believed to initiate the desenitization process. The first specific aim will determine the region of the carboxyl tail involved in agonist-induced down-regulation by truncation analysis. From preliminary results it is evident that a region between s355 and e359 is important. Deletion and mutations will distinguish the motif which triggers the internalization and degradation of the receptor. Additionally, the role the carboxyl tail plays in desensitization will be evaluated. Since phosphorylation is believed to be the instigator of agonist-induced down-regulation and desensitization, the kinases which facilities these processes will be identified. Overall, the proposed project will establish the molecular regulation of ther mu-opioid receptor.